This application is focused on the study of a novel neuroreceptor we have termed to sigma3 receptor. We have designed a series of phenylamino- substituted tetrahydro-naphthalene (phenylaminotetralin, PAT) ligands and radioligands that have very high affinity (e.g., 50 pM) and selectivity for activation of sigma3 receptors that lead to changes in brain catecholamine nerve terminal function. We propose to elucidate recognition and functional characteristics of the sigma3 receptor that should lead to a better understanding of its role in the central nervous system and neurodegeneration. Critical tools for this work are the selective, high affinity PAT-type sigma3 ligands and radioligands we have developed. [3H]PATs will be used in radioreceptor studies with rat and monkey brain homogenates to unequivocally establish that the sigma3 site is not any known receptor. The affinity of a wide range of CNS receptor- active ligands in competition for [3H]PAT-labeled sigma3 sites will be determined to characterize the pharmacological profile of the sigma3 receptor and screen for candidate endogenous sigma3 ligands. The PAT binding pharmacophore at sigma3 receptors will be established by characterizing the affinity, selectivity, and structure-activity requirement (SAR) of PATs and other tetrahydronaphthalenes for [3H]PAT- labeled sigma3 sites. Localization of [3H]PAT-labeled sigma3 receptors in mammalian brain will be determined using quantitative receptor autoradiography in rat, guinea pig, and monkey brain slices. Preliminary data suggest that trans-PATs can potently stimulate striatal dopamine synthesis in vitro and after in vivo administration in rats, while cis- PATs can block this effect. Additional studies will be undertaken using rat brain to establish the PAT agonist and antagonist functional pharmacophore at sigma3 receptors that modulate catecholamine synthesis, and perhaps release, in caudate and other catecholaminergic terminal fields. Selected PAT sigma3 ligands will be screened in vitro for functional effects on catecholamine synthesis by measuring tyrosine hydroxylase activity in rat brain minces. In vivo sigma3 receptor- mediated effects on synthesis and release of 3,4-dihydroxyphenylalanine (DOPA) and catecholamines will be measured in specific brain regions by HPLC-EC, after administering PATs by IP and ICV injection and by intracerebral microdialysis in rats. Post-mortem accumulation of neurotoxic quinone metabolites will be documented in specified brain regions by UV/Vis spectrophotometry. Nerve terminal integrity will be assessed by quantitative autoradiographic assessment of catecholamine neurotransporter density. Additional PAT derivatives to be synthesized will examine: 1) cis vs. trans stereochemical requirement, 2) requirements of the N-binding domain; 3) conformational orientation of the phenethylamine and 1-phenyl moieties; and 4) substitution on the tetrahydronaphthalene and 1-phenyl rings. Enantiomeric resolution of highly active PATs will be undertaken to further examine stereochemical requirements of sigma3 receptor activation. A preliminary sigma3 receptor model has been established that aligns low energy conformations of PATs and non-PATs. Neuropharmacological studies will provide quantitative data for molecular mechanics-based development of a PAT sigma3 receptor model that will be useful in understanding the role of sigma3 receptors to affect brain catecholaminergic nerve terminal function and in neurodegeneration.